Rene S. Hendriksen RSHE@food.dtu.dk +45 72 34 62 88

Antimicrobials, antimicrobial resistance and antimicrobial susceptibility testing

The basics

CRL Training course in AST

Copenhagen, Denmark 23 - 27th Feb. 2009

What is an antibiotic?

Antibiotic is a substance produced by a microorganism,

that have the capacity, in dilute solution, to selectively

inhibit or kill other microorganisms (Paul Vuillemin

1941).

Note the minimum effects on eucaryotic cells

Some of the most toxic substances in the world0.001 – 4 mg/L

Definition of an antimicrobial agent

Antimicrobial agents is a broader term -

Referring to any substance that can affect

microbial life, including synthetic and

semi-synthetic compounds and

substances without selective toxicity (e.g.

disinfectants)

Origin of antimicrobial classes

Class Origin Activity

Aminoglycosides Streptomyces, Micromonospora sp Bactericidal

Cephalosporins Cephalosporium sp Bactericidal

Macrolides Various Actinomycetes Bacteriostatic

Penicillins Penicillium sp Bactericidal

Phenicols Streptomyces venezuelae* Bacteriostatic

Quinolones Synthetic Bactericidal

Rifamycins Amycolatopsis mediterranei Bactericidal

Sulfonamides Synthetic Bacteriostatic

Tetracyclines Streptomyces sp Bacteriostatic

Bactericidal vs. Bacteriostatic antimicrobials

Anti metabolittesSulfonamidesTrimethroprim

Cell wall synthesisβ-lactams:PenicillinCephalosporinsMonobaktamsCarbapenems

Vancomycin

Cell membranePolymyxinAmphotericin

Protein synteseInhibitors (50s)MacrolideschloramphenikolClindamycin

Protein synteseInhibitors (30s)TetracyclinesAminoglykocidesFucidinic acid

DNA replicationFluoquinolonesMetronidazole

RNA-polymeraseRifampicin

Targets of antimicrobial action

Modes of antimicrobial action

• Inhibition of cell wall synthesis

• Inhibition of DNA synthesis

• Inhibition of protein synthesis

• Inhibition of RNA synthesis

• Inhibition of folic acid synthesis

• DNA breakage

• Disruption of osmotic integrity

Note - A limited number of targets

What is antibiotic resistance?

Microbiological definition:

– Resistance is the property of a bacterial strain to

survive at higher antibiotic concentrations compared

with most other members of the same species

(Wildtypes)

Clinical definition:

– Resistance is the ability of a bacterial strain to survive

antimicrobial therapy

Resistance due to a structural or functional trait allowing

tolerance by all members of a bacterial group (species,

genus or even larger group)

– Low affinity of the target

– Impermeability

– Active exporters

– Enzymatic degradation

What is intrinsic resistance?

Example of intrinsic resistanceCefotaxime susceptibility in

E. coli and Acinetobacter baumannii

Mutation (endogenous, vertical)

Gene transfer (exogenous, horizontal)– Transformation (free DNA)– Transduction (with phages)– Conjugation (plasmids – active process)

Two types of acquired resistance

Resistance to two related (avoparcin / vancomycin) or

unrelated drugs (erythromycin / lincosamides) is due to a

single biological mechanism

What is cross-resistance

Mechanisms of resistance• Inactivation of the drug

– Penicillins, aminoglycosides

• Target modification– Tetracyclines, fluoroquinolones

• Drug trapping or titration - hyperproducers– Penicillins, sulphonamides

• Impermeability– Broad range of antibiotics (only reduced susceptibility)

• Active efflux– Disinfectants, metals

How do we measure antimicrobial susceptibility

Agar diffusion method– Disk (tablet) methods– E-test (quantitative)

Dilution methods– Micro broth dilution - Liquid media (quantitative)– Macro broth dilution - Liquid media (quantitative)– Agar dilution - Solid media (quantitative)

MIC

MIC-value

Inhibition zone (mm)

MIC and inhibition zones should correllate for eachantimicrobial for both slow growing and fast growingbacteria

Correllation between MIC and DD

GuidelinesInternational standards describe the methods (DD and MIC) in detail: media, inoculum, incubation, etc. - e.g. CLSI (Clinical and Laboratory Standards Institute)

– CLSI has defined QC reference strains and corresponding the acceptable QC results

– CLSI recommends clinical breakpoints for interpretation

Resistant: Treatment failure can be expected

Sensitive: Succesful treatment can be expected

Intermediary: Treatment is possible if the infection is in bodysites where the antimicrobial is concentrated. Primarily a bufferzone to avoid misinterpretation

Defined by EUCAST (The European Committee on Antimicrobial Susceptibility Testing – www.eucast.org)

- for monitoring purposeonly, no relation to clinical data

- defined by the wild type distribution of the bacteria

- all isolates with MICsabove the WT distribution are calledresistant

Epidemiological Cut Off Values

Techniques - Pros and Cons

MIC determination

– Golden standard for AST– Data more reproducible– Better separation of R/S– More information– Expensive– Only pure cultures– Contaminations more

difficult to detect

Diffusion techniques

– Cheaper– Primary material– See contaminations– Quick screening (4 hours)– Qualitative information– Less reproducible data– Standardisation more

difficult

Disk diffusion - Considerations

Disk diffusion methods gives reliable and reproducible data, if standardization and quality assurance is used

• Use international guidelines (e.g. CLSI)

• Standardize the method (inoculum, media, incubation, reading

of results)

• Use ring tests (define criteria for acceptance, evaluate results)

• Use reference strains for method validation

• Act on deviations on the QC-strain

• Document corrective actions

StandardisationAll methods are extremely sensitive to variations in performance

Factors that influence the result:

- Size of inoculum- Contents and acidity (pH) of the broth or agar- Incubation time and temperature- Reading procedures

Moreover, for the diffusion methods:- Diffusion rate of the antimicrobial into the agar- Depth of the agar- Dryness of the agar- Growth rate of the bacteria

Thank you!

Rene Hendriksen (rshe@food.dtu.dk)